Accepted Manuscript

Synthesis and anti-parasitic activity of a novel quinolinone-chalcone series

Marina Roussaki, Belinda Hall, Sofia Costa Lima, Anabela Cordeiro da Silva,

Shane Wilkinson, Anastasia Detsi

PII: S0960-894X(13)01121-9

DOI: http://dx.doi.org/10.1016/j.bmcl.2013.09.047

Reference: BMCL 20899

To appear in: Bioorganic & Medicinal Chemistry Letters

Received Date: 6 August 2013

Revised Date: 13 September 2013

Accepted Date: 16 September 2013

Please cite this article as: Roussaki, M., Hall, B., Lima, S.C., da Silva, A.C., Wilkinson, S., Detsi, A., Synthesis and

anti-parasitic activity of a novel quinolinone-chalcone series, Bioorganic & Medicinal Chemistry Letters (2013),

doi: http://dx.doi.org/10.1016/j.bmcl.2013.09.047

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1

Synthesis and anti-parasitic activity of a novel quinolinone-

chalcone series

Marina Roussaki,a Belinda Hall,b Sofia Costa Lima,c Anabela Cordeiro da Silva,c,d

Shane Wilkinson,b Anastasia Detsia*

a Laboratory of Organic Chemistry, School of Chemical Engineering, National

Technical University of Athens, Zografou Campus 15780 Athens, Greece

bSchool of Biological and Chemical Sciences, Queen Mary University of London,

England

cParasite Disease Group, IBMC-Institute for Molecular and Cell Biology, University of

Porto, Rua do Campo Alegre, 823, 4150-180, Porto, Portugal

dDepartment of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de

Jorge Viterbo Ferreiera, 228, 4050-313 Porto, Portugal

* Corresponding author

e-mail: adetsi@chemeng.ntua.gr

Tel.: +302107724126

Fax: +302107723072

Abstract

A series of novel quinolinone-chalcone hybrids and analogues were designed,

synthesized and their biological activity against the mammalian stages of

Trypanosoma brucei and Leishmania infantum evaluated. Promising molecular

scaffolds with significant microbicidal activity and low cytotoxicity were identified.

Quinolinone-chalcone 10 exhibited anti-parasitic properties against both organisms,

being the most potent anti-L.infantum agent of the entire series (IC50 value of 1.3 ±

0.1 μM). Compounds 4 and 11 showed potency toward the intracellular, amastigote

stage of L. infantum (IC50 values of 2.1 ± 0.6 and 3.1 ± 1.05 μM, respectively).

Promising trypanocidal compounds include 5 and 10 (IC50 values of 2.6 ± 0.1 and 3.3

± 0.1 μM, respectively) as well as 6 and 9 (both having IC50 values of <5 μM).

Chemical modifications on the quinolinone-chalcone scaffold were performed on

2

selected compounds in order to investigate the influence of these structural features

on antiparasitic activity.

Keywords

Quinolinone-chalcones, antiparasitic activity, T. brucei, L. infantum, cytotoxicity

Diseases caused by protozoan parasites belonging to the genera Trypanosoma

and Leishmania are responsible for high mortality and morbidity each year,

particularly in low-income countries.1 With no immediate prospect of a vaccine, drugs

are the only option to treat these infections. The use of current therapies is

controversial as many are toxic, some are mutagenic while clinical resistance is on

the rise. Additionally, many require medical supervision while being administered

which all contribute to the cost per treatment. Against this backdrop, there is an

urgent requirement for new, safe, cheap drugs. However, as drug development is

expensive, the development of new chemotherapies targeting trypanosomatid

parasites is perceived as not being commercially attractive. As a result, trypanosomal

and leishmanial diseases are said to be neglected due to the lack of funds available

to combat these pathologies.

Human African trypanosomiasis (HAT) is prevalent in sub-Saharan Africa with an

estimated 50 million people living in areas deemed at risk.2 This life-threatening

zoonosis is caused by Trypanosoma brucei, which is transmitted to humans by the

blood feeding habits of its insect vector, the tsetse fly.3,4 Due to vector control

programmes coupled with improved public health surveillance, the number of

infections have fallen dramatically over the last 15 years from a peak estimated

between 400,000 - 500,000 in 1998 to less than 30,000 cases in 2009.5,6 However,

previous epidemics have shown that unless such measures are maintained, HAT can

readily flare up causing a rapid increase in prevalence following political and

socioeconomic disorder, potentially resulting in the deaths of tens of thousands of

people.6,7

Leishmaniasis is endemic in many tropical and sub-tropical regions, with

approximately 350 million people living in “at risk” areas. Estimates indicate that 12

million people currently suffer from leishmaniasis, with about 2 million new cases and

50,000 deaths occurring each year.2,8 More than 20 human infectious Leishmania

species are responsible for a complex set of pathologies that range from a self

3

limiting cutaneous form to a potentially fatal visceral disease. The parasite is

transmitted through the hematophagous feeding habits of pregnant, female sandflies

belonging to the subfamily Phlebotominae. The main transmission cycle is zoonotic

although anthroponotic routes are now common for some species, especially in an

urban context. However, due to intravenous drug usage/needle sharing, transfusion

with infected blood and other blood-borne contact coupled with global warming and

the spread of the insect vector, leishmaniasis is beginning to emerge as a problem in

non-endemic regions.

Nature has provided an inspiration for the design and synthesis of many

compounds, providing novel scaffolds that have been exploited in drug development.

Two groups of molecules that have shown potential in this regard are the chalcones

and quinolinones. Chalcones are open chain flavonoids, comprised of two aryl rings

(rings A and B) linked by an α,β-unsaturated carbonyl moiety (Figure 1). They are

synthesized predominantly by plants and are important precursors in a number of

biochemical pathways ranging from flower pigmentation to biological defence against

phytopathogens and insects.9

(Figure 1)

Due to their distribution in edible plants and to their powerful and diverse

biological activities, chalcones have been intensively investigated as scaffolds in the

development of anti-cancer,10 antioxidant,11,12 anti-angiogenic,13 and anti-

inflammatory14,15 agents. Several natural and synthetic chalcones have been shown

to possess anti-parasitic activities such as licochalcone A (Figure 2), a natural

prenylated chalcone isolated from the Chinese licorice, identified as a potent anti-

leishmanial agent which acts by inhibiting fumarate reductase in L. major and L.

donovani,16,17 and hybrid scaffolds incorporating the chalcone moiety, which are

emerging as potent lead structures targeting Leishmania and Trypanosoma

species.18-20

The quinolinone moiety (Figure 1) is a common structural motif found in numerous

quinolinone alkaloid natural products.21-23 Quinolinone analogues possess important

biological properties such as antioxidant,24,25 anti-inflammatory25,26 and enzyme-

inhibitory activity.27 As with the chalcones, several quinolinone-based compounds

have emerged as anti-parasitic lead structures, notably the natural products

dictyolomide A and B (Figure 2), which possess leishmanicidal activity,28 and 3-

substituted-quinolinone derivatives that target both L. donovani and T. brucei. 29

4

(Figure 2)

As a continuation of our studies toward the synthesis and biological evaluation of

chalcone12 and quinolinone24,25 derivatives, here we present the synthesis of novel

hybrid compounds encompassing the 4-hydroxy-2-quinolinone heterocyclic system

and the chalcone moiety in one molecular scaffold in order to investigate the potential

synergistic effect of these two pharmacophores against the protozoan parasites T.

brucei and L. infantum. Our approach substitutes ring A of the chalcone moiety with

the 4-hydroxy-2-quinolinone structure. The literature concerning the synthesis and

biological evaluation of this type of compounds is scarce and, to our knowledge, this

is the first time that their anti-parasitic activities against T. brucei and L. infantum

have been reported.

The synthesis of the desired compounds was accomplished via an aldol

condensation reaction between 3-acetyl-4-hydroxy-2-quinolinones and variably

substituted benzaldehydes. The required starting material, 3-acetyl-4-hydroxy-2(1H)-

quinolinone (3), was synthesized using our previously developed methodology which

includes C-acylation of ethyl acetoacetate by 2-methyl-3,1-benzoxazin-4-one (1) and

subsequent cyclization of the C-acylation product 2 in basic conditions (Scheme 1).30

(Scheme 1)

The methodologies reported in the literature for the synthesis of quinolinone-

chalcone hybrids are limited: Kalechits et al. have prepared a series of quinolinone-

chalcone analogues by refluxing 3-acetyl-4-hydroxy-2(1H)-quinolinone with various

benzaldehydes in pyridine using drops of piperidine as catalyst.31 In 2006 a

microwave-assisted synthesis of this type of compounds in EtONa/EtOH was

reported32 whereas the most recent methodology (2011) involves a solvent-free

protocol using silica sulfate catalyst which efficiently performs the crossed-aldol

reaction required.33

In the present study, various conditions were investigated in order to synthesize

the target quinolinone-chalcone derivatives. Optimization of the reaction conditions

was performed using 3-acetyl 4-hydroxyquinolin-2(1H)-one (3) and 4-methyl-

benzaldehyde as starting materials and carrying out the reaction using ethanol or

pyridine as solvents and aqueous KOH or piperidine as bases (Table 1).

(Table 1)

5

Optimum results were obtained using catalytic amounts of piperidine in EtOH

(Scheme 2). Using these conditions, the reaction is completed in 1–2 h (monitored by

TLC). Acidification with aqueous HCl (10%), followed by filtration of the precipitating

solid, results to the isolation of the desired products in satisfactory yields (40–60%)

and purity, as confirmed by NMR spectroscopy.

(Scheme 2)

Twelve compounds were synthesized in total, bearing either a variety of electron –

donating (CH3, OCH3, C(CH3)3, OH) or electron – withdrawing substituents (NO2,

CF3, COOH) on ring B of the chalcone framework (compounds 4-13) or an extended

conjugated system (compounds 14 and 15).

In order to further investigate the structure – activity relationship of the series of

quinolinone-chalcone hybrids, we decided to modify two of the most characteristic

structural features of the quinolinone-chalcone framework, namely the amide moiety

and the α,β-unsaturated carbonyl system. In this context, modification of the amide

hydrogen of the heterocyclic ring of compounds 5 and 9 by alkylation was initially

undertaken (Scheme 3). The required starting materials, N-ethyl and N-benzyl-3-

acetyl-4-hydroxy-2-quinolinones 18 and 19, were synthesized via reductive alkylation

of methyl anthranilate by the appropriate aldehyde, acylation of the secondary amine

using 2,2,6-trimethyl-1,3-dioxan-4-one as an acylating agent and cyclization of the

crude product under basic conditions.34 The final alkylated quinolinone-chalcones 20-

23 were prepared via a crossed aldol coupling reaction, according to the initial

method (Scheme 3).

(Scheme 3)

Moreover, pyrazoline analogues of compounds 5 and 9 were synthesized, by

refluxing the corresponding chalcone with phenyl hydrazine or hydrazine hydrate in

glacial acetic acid solution35 (Scheme 4). The final pyrazoline analogues 24 and 25

were obtained after filtration of the solid product formed and the pure compounds

were collected after trituration with methanol in 40% yield.

(Scheme 4)

6

All the 1H NMR spectra of the synthesized chalcones showed the characteristic

signals of the vinylic protons α and β at 6.5-8 ppm, with coupling constants J=12-18

Hz, indicating the E-geometric configuration for all compounds, and a single peak at

17-18 ppm assigned to the enolic proton of position 4 which is involved in a strong

hydrogen bond with the carbonyl oxygen.

The anti-parasitic activities of the compounds synthesized in this work were

evaluated in vitro against bloodstream form Trypanosoma brucei and axenically

cultured intracellular Leishmania infantum amastigotes (Table 2).

(Table 2)

Out of twelve quinolinone-chalcone analogues screened, five were shown to have

growth inhibitory activity against T. brucei. The most potent of these was compound 5

(IC50 = 2.6 ± 0.1 μM) followed by compounds 10, 6, 9 and 7 (decreasing potency),

with all five trypanocidal structures containing electron-donating substituents on ring

B of the chalcone motif. Analysis of data suggests that the position and number of

these groupings all contribute to a compound’s anti-parasitic property. For the most

effective trypanocidal agent, compound 5, the electron-donating methyl substituent is

located at the 2-position on ring B while its isomer, compound 4 that contains the

methyl grouping at the 4-position, had no effect on trypanosomal growth at

concentration up to 10 μM. Similarly, compound 7, which contains a single methoxy

at the 3-position on ring B is trypanocidal (IC50 value of 6.5 ± 0.1 μΜ) while the

isomeric chalcone 8, that contains a 4-methoxy group, had no effect on parasite

growth. The presence of two electron-donating substituents was shown to lead to a

slight increase in trypanocidal activity: compound 9 which contains two methoxy

groups at the 3- and 4- positions on ring B generated an IC50 value of 4.9 ± 0.2 μΜ

while an IC50 value of 4.9 ± 0.1 μΜ was displayed by compound 6 that contains a

methoxy and hydroxyl at the 3- and 4-positions, respectively. The remaining

trypanocidal chalcone, compound 10, possesses an ortho-(di-tert-butyl)-phenol

substitution. Despite being the second most potent anti-trypanosomal structure of the

chalcone series (IC50 value of 3.3 ± 0.1 μΜ), it displayed high cytotoxicity (IC50 value

26 μΜ).

For compounds 5 and 9, analogues containing an alkyl substituent on the amidic

nitrogen present on the heterocyclic ring of the quinolinone moiety (compounds 20-

7

23) were synthesized. N-ethyl-analogues 20 and 21 did not exhibit growth inhibitory

properties against bloodstream form T. brucei. Compounds 22 and 23, the N-benzyl

analogues of chalcones 5 and 9, respectively, showed lower activity against T. brucei

than the non-alkylated compounds, albeit higher than the N-ethyl analogues. These

results suggest that the hydrogen of the heterocyclic amide group is important in the

mechanism of action of these compounds. Likewise, alterations to the quinolinone-

chalcone structure at other sites, by incorporating electron withdrawing groups

(COOH, CF3 and NO2) on ring B of the chalcone (compounds 11, 12, and 13) or

extending the conjugation system between the quinolinone and chalcone motifs

(compounds 14 and 15), had no effect on trypanosomal growth at concentration up to

10 μM.

Modifying the α,β-unsaturated carbonyl system by synthesizing the pyrazoline

analogues 24 and 25, resulted to a remarkable increase in antiparasitic activity

against bloodstream form T. brucei: both compounds exhibited IC50 lower than the

reference drug nifurtimox thus are the most active against T. brucei parasites among

all the 18 compounds tested in this work. Pyrazoline 24 (IC50 value of 1.46 ± 0.1) can

be regarded as a promising lead antiparasitic compound as it is also not cytotoxic

against THP1 cells.

When the quinolinone-chalcone hybrid series were screened against the

intracellular amastigote stage of L. infantum, eleven structures (compounds 4-15, 20

and 21) were shown to affect parasite growth. The most potent of these were

compounds 10, 4 and 11 which yielded IC50 values of 1.3 ± 0.1, 2.1 ± 0.6 and 3.1 ±

1.0 µM, respectively. Intriguingly, the structure activity relationships observed using

this compounds series against L. infantum were markedly distinct from those noted

when targeting T. brucei, with many of the differences being directly opposite. For

example, moving the methyl substituent from 4-position (compound 4) to 2-position

(compound 5) or the methoxy group from the 4-position (compound 8) to 3-position

(compound 7) resulted in a significant reduction in leishmancidal activity in contrast to

the situation noted for T. brucei (Table 2). Moreover, the electronic nature of the

substituents does not seem to play a very important role in the anti-leishmanial

activity as it did for T. brucei: compound 11 bearing an electron-withdrawing

trifluoromethyl group (a methyl group isostere) at the 4-position of ring B is the third

more active agent of the series against L. infantum with the NO2 and COOH

containing derivatives (compounds 12 and 13) having an affect against L. infantum

amastigotes. Why these conflicting SAR differences arise is unclear. This could

8

reflect that these parasites reside at different locations within the mammalian host: T.

brucei is an extracellular pathogen found in the hosts’ bloodstream whereas L.

infantum can invade and grow in host macrophages, residing within an acidic

compartment. Alternatively, this could simply be due to the concentration ranges

used in the screens: many leishmanicidal compounds have IC50 values >10 µM, the

maximum level used against T. brucei.

N-alkylation of the heterocyclic amide group resulted in compounds with lower or

slightly better antileishmanial activity as compared to the non-alkylated analogues,

indicating that the N-H group is probably crucial for anti-leishmanial activity as it was

also in the case of anti-trypanosomal activity. Pyrazoline 24 showed lower activity

against L. infantum in comparison with the chalcone analogue 9 whereas pyrazoline

25, an analogue of chalcone 5, was the most active anti-leishmanial agent among all

the compounds tested exhibiting IC50 value of 0.71 μM. It is worth noting that

pyrazoline 25 showed the best antiparasitic activity against both parasites.

To evaluate their effects on mammalian cells, cytotoxicity assays were performed

with all compounds against differentiated THP-1 macrophages (Table 2). Of the most

potent agents (IC50 values <10 μM), the compounds 4, 5, 7, 9, 11 and 24 had a no

growth-inhibitory effect on THP-1 cells at concentrations at 30 M (5, 7 and 9) or 50

M (4, 11 and 24) whereas compounds 6 and 10 had IC50 values of around 20 μM.

Exact IC50 cytotoxicity data could not be determined due to the issues relating to

compound solubility in DMSO and other common solvents. Comparison of potency

against the parasite and mammalian lines indicates that pyrazoline 24 and chalcone

5 are the most effective trypanocidal agents while chalcone analogues 4 and 11 are

interesting lead structures targeting L. infantum.

Studies concerning the actual mechanism of action of chalcones against

kinetoplastids are still underway by several research groups. For instance, Chen et

al.17 have shown that chalcones active against Leishmania major and L. Donovani,

inhibit the action of fumarate reductase (FRD), one of the enzymes of the parasite

respiratory chain which is not present in mammalian cells, and suggested that this

enzyme might be the specific target for the chalcones. However, fumarate reductase

activity does not appear to be essential in the bloodstream form of T. brucei, so other

mechanisms must be involved in cytotoxicity towards these parasites.36 Recently,

9

several chalcones have been shown to induce production of ROS and trigger

apoptosis in cancer cells37 and that this kind of pathway is present in kinetoplastids.38

The role of the quinolinone moiety is unclear at this stage and no reports

concerning the antiparasitic activity of quinolinone-chalcone hybrids or the

corresponding pyrazoline analogues are available, therefore we can only speculate

that our compounds could follow either of the above mentioned mechanisms. These

studies could be the subject of further investigation.

The emphasis of this work was the synthesis of novel hybrid compounds,

encompassing the quinolinone and the chalcone moiety in one molecular scaffold. A

series of these analogues, bearing a wide variety of substituents were synthesized

and their in vitro anti-parasitic activity against bloodstream form T. brucei and the

intracellular L. infantum amastigotes, as well as their cytotoxicity against mammalian

cells, were evaluated. Several compounds elicited growth inhibitory activity against

one or both parasites, promoting further investigations into deciphering any structure

activity relationships. Of this series, the most potent structure against T. brucei was

compound 5 (IC50 value of 2.6 ± 0.1 μM) followed by compounds 6 and 9 (IC50 values

of <5 μM), while compounds 4 and 11 showed potential as leishmanidal agents (IC50

values of 2.1 ± 0.6 and 3.1 ± 1.0 μM, respectively). None of these lead structures

displayed cytotoxicity towards the mammalian THP-1 cell line. In contrast chalcone

10, although active against both parasites (and the most potent against L. infantum),

did display toxicity toward mammalian cells. We were able to show that substituents

on ring B of the chalcone motif and the amidic nitrogen present on the heterocyclic

ring of the quinolinone moiety are important determinates in mediating the biological

activities of these compounds. These features can be incorporated into the next

generation of quinolinone-chalcone hydrid scaffolds with the goal of developing new,

safe, cost effective treatments targeting these neglected tropical diseases. Structural

modification of the α,β-unsaturated carbonyl system via transformation to the

corresponding pyrazoline analogues, resulted to compounds 24 and 25 with

remarkable in vitro anti-parasitic activity against bloodstream form T. brucei. Although

pyrazoline 25 exhibited the best activity against both parasites and is the most potent

among all the compounds tested in this work, its high cytotoxicity againt mammalian

cells prohibits its further consideration as a lead compound. On the other hand,

pyrazoline 24 should be considered as a promising lead trypanocidal agent as it

possesses high antiparasitic activity and is not cytotoxic. To our knowledge the

pyrazoline scaffold, although extensively studied for other biological acitivities, has

10

not been yet explored as a structural feature for the development of antiparasitic

agents.

Ackowledgements

This collaboration has been promoted by the COST Action CM0801 “New drugs for

neglected diseases”.

Supplementary data

Supplementary data associated with this article can be found, in the online version,

at……..

References

1. “Sustaining the drive to overcome the global impact of neglected tropical

diseases”, Second WHO report on neglected tropical diseases, Crompton D.

WT, Ed., 2013, WHO reference number: WHO/HTM/NTD/2013.1

2. Stuart, K.; Brun, R.; Croft, S.; Fairlamb, A.; Gurtler, R.E.; McKerrow, J.; Reed,

S.; Tarleton, R., J. Clin. Invest. 2008, 118, 1301.

3. Sokolova, A.Y.; Wyllie, S.; Patterson, S.; Oza, S.L.; Read, K.D.; Fairlamb, A.H.,

Antimicrob. Agents Chemoth. 2010, 54, 2893.

4. Kennedy, P.G.E., J. Neurol. 2006, 253, 411.

5. Simarro, P. P.; Jannin, J.; Cattand, P., PLoS Med. 2008, 5, 55.

6. Brun, R.; Blum, J.; Chappuis, F.; Burri, C., Lancet 2010, 375, 148.

7. Steverding, D., Parasites and Vectors 2008, 12, 1, 3.

8. Croft, S. L.; Coombs, G. H., Trends in Parasitol. 2003, 19, 502.

9. Batovska, D.I.; Todorova, I., Curr. Clin. Pharmac. 2010, 5, 1.

10. Roman, B. I.; Heugebaert, T. S. A.; Bracke, M. E.; Stevens, C. V. Curr. Med.

Chem. 2013, 20, 186.

11. Shenvi, S.; Kumar, K.; Hatti, K. S.; Rijesh, K.; Diwakar, L.; Reddy, G. C. Eur. J.

Med. Chem. 2013, 62, 435.

12. Detsi A.; Majdalani M.; Kontogiorgis C.A.; Hadjipavlou-Litina D.; Kefalas P.,

Bioorg. Med. Chem. 2009, 17, 8073.

13. Mojzis, J.; Varinska, L.; Mojzisova, G.; Kostova, I.; Mirossay, L., Pharmacol.

Res. 2008, 57, 259.

14. Nowakowska, Z., Eur. J. Med. Chem. 2007, 42, 125.

15. Bano, S.; Javed, K.; Ahmada, S.; Rathish, I.G.; Singh, S.; Chaitanya, M.;

Arunasree, K.M.; Alam, M.S. Eur. J. Med. Chem. 2013, 65, 51.

11

16. Kayser, O.; Kiderlen, A.F.; Croft, S.L., Parasitol. Res. 2003, 90, S55.

17. Chen, M.; Zhai, L.; Christensen, S. B.r; Theander, T. G.; Kharazmi, A.

Antimicrob. Agents Chemoth. 2001, 45, 2023.

18. Ryczak, J.; Papini, M.; Lader, A.; Nasereddin, A.; Kopelyanskiy, D.; Preu, L.;

Jaffe, C.L.; Kunick, C. Eur. J. Med. Chem. 2013, 64, 396.

19. Tyagi, V.; Khan, S.; Shivahare, R.; Srivastava, K.; Gupta, S.; Kidwai, S.;

Srivastava, K.; Puri, S. K.; Chauhan, P. M. S. Bioorg. Med. Chem. Lett. 2013,

23, 291.

20. Qiao, Z.; Wang, Q.; Zhang, F.; Wang, Z.; Bowling, T.; Nare, B.; Jacobs, R.T.;

Zhang, J.; Ding, D.; Liu, Y., J. Med. Chem. 2012, 55, 3553.

21. He, J.; Lion,U.; Sattler,I.; Gollmick, F.A.; Grabley,S.; Cai, J.; Meiners, M.;

Schunke, H.; Schaumann, K.; Dechert,U.; Krohn, M. J. Nat. Prod. 2005, 68,

1397.

22. Nakashima, K.; Oyama, M.; Ito, T.; Akao, Y.; Witono, J.R.; Darnaedi, D.;

Tanaka, T.; Murata, J.; Iinuma, M. Tetrahedron 2012, 68, 2421.

23. Gao, H.; Zhang, L.; Zhu, T.; Gu, Q.; Li, D. Chem. Pharm. Bull. 2012, 60, 1458.

24. Angeleska, S.; Kefalas, P.; Detsi, A. Tetrahedron Lett. 2013, 54, 2325.

25. Detsi A.; Bouloumbasi D.; Prousis K.C.; Koufaki M.; Athanasellis G.; Melagraki

G.; Afantitis A.; Igglessi-Markopoulou O.; Kontogiorgis C.; Hadjipavlou-Litina

D.J., J. Med. Chem. 2007, 50, 2450-2458.

26. Jonsson, S.; Andersson, G.; Fex, T.; Fristedt, T.; Hedlund, G.; Jansson, K.;

Abramo, L.; Fritzson, I.; Pekarski, O.; Runstrom, A.; Sandin, H.; Thuvesson, I.;

Bjork, A. J. Med. Chem. 2004, 47, 2075.

27. Larsson, E.A.; Jansson, A.; Ng, F.M.; Then, S.W.; Panicker,R.; Liu, B.;

Sangthongpitag, K.; Pendharkar, V.; Tai, S.J.; Hill,J.; Dan,C.; Ho, S.Y.; Cheong,

W.W.; Poulsen,A.; Blanchard, S.; Lin, G.R.; Alam,J.; Keller,T.H.; Nordlund, P. J.

Med. Chem. 2013, 56, 4497.

28. Lavaud C.; Massiot G.; Vasquez C.; Moretti C.; Sauvain M.; Balderrama L.,

Phytochemistry 1995, 40, 317.

29. Audisio D.; Messaoudi S.; Cojean S.; Peyrat J.F.; Brion J.D.; Bories C.; Huteau

F.; Loiseau P.M.; Alami M., Eur. J. Med. Chem. 2012; 52; 44-50.

30. Detsi A.; Bardakos V.; Markopoulos J.; Igglessi-Markopoulou O., J. Chem. Soc.

Perkin Trans. 1996, 1, 2909.

31. Kalechits G.V.; Olkhovik V.K.; Kalosha I.I.; Skakovskii E.D.; Pap A.A.; Zenyuk

A.A.; Matveenko Y.V., Russ. J. Gen. Chem. 2001, 71, 1257.

32. Sarveswari, S.; Raja, T. K., Ind. J. Heterocyclic Chem., 2006, 16, 171.

33. Bhupathi, Raja S.; Devi, B. Rama; Dubey, P. K. Asian J. Chem. 2011, 23, 4215.

12

34. Boyle R.G.; Imogai H.J.; Cherry M.; Humphries A.J; Navarro E.F.; Owen D.R.;

Dales N.A.; Lamarche M.; Cullis C., Gould A.E. WO 2005/028474 A2

35. Sivakumar P.M.; Ganesan S.; Veluchamy P.; Doble M., Chem. Biol. Drug. Des.,

2010, 76, 407.

36. Alsford, S.; Turner, D. J.; Obado, S. O.; Sanchez-Flores, A.; Glover, L.;

Berriman, M.; Hertz-Fowler, C.; Horn, D. Genome Res., 2011, 21, 915.

37. Wu, W.; Ye, H.; Wan, L.; Han, X.; Wang, G.; Hu, J.; Tang, M.; Duan, X.; Fan, Y.;

He, S.; Huang, L.; Pei, H.; Wang, X.; Li, X.; Xie, C.; Zhang, R.; Yuan, Z.; Mao,

Y.; Wei, Y.; Chen, L., Carcinogenesis, 2013, 34, 1636.

38. Piacenza, L.; Irigoin, F.; Alvarez, M. N.; Peluffo, G.; Taylor, M. C.; Kelly, J. M.;

Wilkinson, S. R.; Radi, R., Biochem. J., 2007, 403, 323.

13

Captions of Figures, Schemes and Tables

Figure 1. General structure of the chalcone and quinolinone molecular scaffolds.

Figure 2. Quinolinone and chalcone analogues with antiparasitic activity

Scheme 1. Synthesis of the starting 3-acetyl-4-hydroxy-2(1H)-quinolinone (3).

Reagents and conditions: (i) (CH3CO)2O, 130oC, 2h; (ii) CH3COCH2COOEt, t-BuOK,

t-BuOH, r.t.; (iii) aq. Na2CO3/NaOH, r.t.

Scheme 2: Synthesis of the target compounds. Reagents and conditions: (i)

piperidine(cat.), EtOH, reflux

Scheme 3. Synthetic approach to N-alkylated quinolinone-chalcones. Reagents and

conditions: (i) RCHO, H-B(OAc)3Na, CH3COOH, CH2Cl2, r.t.; (ii) 2,2,6-trimethyl-1,3-

dioxan-4-one, toluene, reflux;(iii) EtOH, EtONa, reflux;(iv) piperidine (cat.), EtOH,

reflux

Scheme 4. Synthetic approach to pyrazoline analogues 24, 25. Reagents and

conditions: (i) PhNHNH2 (for compound 24) or H2NNH2.H2O (for compound 25),

CH3COOH, reflux

Table 1. Optimization of reaction conditions for the synthesis of quinolinone-

chalcones

Table 2: Anti-parasitic and cytotoxic activities of compounds 4-15 and 20-25

14

Figure 1. General structure of the chalcone and quinolinone molecular scaffolds.

15

Figure 2. Chalcone and quinolinone analogues with antiparasitic activity

16

Scheme 1. Synthesis of the starting 3-acetyl-4-hydroxy-2(1H)-quinolinone (3).

Reagents and conditions: (i) (CH3CO)2O, 130oC, 2h; (ii) CH3COCH2COOEt,

t-BuOK, t-BuOH, r.t.; (iii) aq. Na2CO3/NaOH, r.t.

17

Scheme 2: Synthesis of the target compounds. Reagents and conditions: (i)

piperidine(cat.), EtOH, reflux

18

Scheme 3. Synthetic approach to N-alkylated quinolinone-chalcones. Reagents

and conditions: (i) RCHO, H-B(OAc)3Na, CH3COOH, CH2Cl2, r.t.; (ii) 2,2,6-

trimethyl-1,3-dioxan-4-one, toluene, reflux;(iii) EtOH, EtONa, reflux;(iv) piperidine

(cat.), EtOH, reflux

19

Scheme 4. Synthetic approach to pyrazoline analogues 24, 25. Reagents and

conditions: (i) PhNHNH2 (for compound 24) or H2NNH2.H2O (for compound 25),

CH3COOH, reflux

20

Table 1. Optimization of reaction conditions for the synthesis of quinolinone-chalcone

hybrids

Base Solvent Yield (%)

1eq 1eq KOH(5eq) EtOH 18

1eq 1eq Piperidine(1.2eq) EtOH <10

1eq 1eq Piperidine(1.2eq) Pyridine <10

1eq 1eq Piperidine(cat.) EtOH 49

21

Table 2: Anti-parasitic and cytotoxic activities of compounds 4-15 and 20-25

Compound IC50 (µM)

T.brucei L. infantum THP1

4 >10 2.1 ± 0.6 >50a

5 2.6 ± 0.1 >50 >50a

6 4.9 ± 0.1 11.5 ± 1.7 23.3 ± 1.4a

7 6.5 ± 0.1 28.4 ± 3.5 38.5 ± 2.1a

8 >10 12.7 ± 3.0 >50a

9 4.9 ± 0.2 7.5 ± 2.7 >50a

10 3.3 ± 0.1 1.3 ± 0.1 26.2 ± 2.7 a

11 >10 3.1 ± 1.0 >50a

12 >10 26.9 ± 4.6 >50a

13 >10 18.4 ± 6.2 >50a

14 >10 >50 >50a

15 >10 20.0 ± 6.6 >50a

20 >10 24.8 ± 3.9 >50a

21 >10 >50 >50a

22 6.17±0.7 >25 45.7 ± 3.1 a

23 5.68±0.5 >25 28.7 ± 1.7 a

24 1.46±0.1 13.46±0.61 >50a

25 1.43±0.1 0.71±0.07 4.15±0.19

Nifurtimox 2.9 0.3 - >100b

Amphotericin B - 1.2 0.1 23.8 2.3c

a Performed using the MTT assay. b The cytotoxicity of nifurtimox toward

differentiated THP-1 cells was performed using alamarBlue™. c The cytotoxicity

towards Ampotericin B was established through MTT assay.

Graphical Abstract

Compound IC50 (μM)

T. brucei L. infantum

4 R1

=R2= R

4=H, R

3=CH3 >10 2.1± 0.6

5 R1=CH3, R

2=R

3=R

4=H 2.6 ± 0.1 >50

10 R1=H, R

2=R

4=C(CH3)3,R

3=OH 3.3 ± 0.1 1.3 ± 0.1

Nifurtimox 2.9 0.3 -

Amphotericin B - 1.2 0.1